Optimization of activity, selectivity, and liability profiles in 5-oxopyrrolopyridine DPP4 inhibitors leading to clinical candidate (Sa)-2-(3-(aminomethyl)-4-(2,4-dichlorophenyl)-2-methyl-5-oxo-5H-pyrrolo[3,4-b] pyridin-6(7H)-yl)-N, N-dimethylacetamide (BMS-767778)

Article abstract of DOI:10.1021/jm4008906

Optimization of a 5-oxopyrrolopyridine series based upon structure-activity relationships (SARs) developed from our previous efforts on a number of related bicyclic series yielded compound 2s (BMS-767778) with an overall activity, selectivity, efficacy, PK, and developability profile suitable for progression into the clinic. SAR in the series and characterization of 2s are described.

Full text of DOI:10.1021/jm4008906

Article
pubs.acs.org/jmc
Optimization of Activity, Selectivity, and Liability Profiles in
5‑Oxopyrrolopyridine DPP4 Inhibitors Leading to Clinical
Candidate (Sa)‑2-(3-(Aminomethyl)-4-(2,4-dichlorophenyl)-2-methyl-
5-oxo‑5H‑pyrrolo[3,4‑b]pyridin-6(7H)‑yl)‑N,N‑dimethylacetamide
(BMS-767778)
Pratik Devasthale,*,† Ying Wang,† Wei Wang,† John Fevig,† JianXin Feng,† Aiying Wang,‡ Tom Harrity,‡
Don Egan,‡ Nathan Morgan,‡ Michael Cap, ‡ Aberra Fura,§ Herbert E. Klei,∥ Kevin Kish,∥
Carolyn Weigelt,⊥ Lucy Sun,§,× Paul Levesque,§ Frederic Moulin,§ Yi-Xin Li,§ Robert Zahler,†,#
Mark S. Kirby,‡ and Lawrence G. Hamann†,∞
‡
§
∥
†Metabolic Diseases Chemistry, Metabolic Diseases Biology, Pharmaceutical Candidate Optimization, Protein Science and
⊥
Structure, Computer-Assisted Drug Design, Bristol-Myers Squibb Research and Development, Princeton, New Jersey 08543-5400,
United States
ABSTRACT: Optimization of a 5-oxopyrrolopyridine series based upon structure−activity relationships (SARs) developed from
our previous efforts on a number of related bicyclic series yielded compound 2s (BMS-767778) with an overall activity, selec-
tivity, efficacy, PK, and developability profile suitable for progression into the clinic. SAR in the series and characterization of 2s
are described.
greater placebo-subtracted HbA1c-lowering (>0.7 versus 0.4
that was achieved with 5 mg dose of saxagliptin) was observed
in a 6-week phase II clinical trial with saxagliptin at 100 mg
(unpublished data). However, because of potential concerns
about using suprapharmacological doses, it was decided not
to explore this dose in the clinic. It was therefore desirable to
identify a potent, selective, and efficacious DPP4 inhibitor, opti-
mally in a novel chemotype, with a high margin of safety to test
the hypothesis in the clinic that greater HbA1c lowering could
be achieved with continuous maximal DPP4 inhibition. This
paper describes the culmination of those efforts with the iden-
tification of 2s (BMS-767778), an optimized candidate from the
5-oxopyrrolopyridine series that was advanced into the clinic.
The design and rationale for arriving at a variety of substituted
bicyclic scaffolds have been recently described6c,8a,8b and are sum-
marized in Figure 1. Earlier work from our laboratories6a−c had
identified the optimal point of attachment of the R group on a
number of these scaffolds as indicated in Figure 1. X-ray struc-
tures from earlier series had suggested that this R group was
exposed to solvent. The rationale for having a nitrogen atom on
these bicyclic cores at this point of attachment was to allow facile
INTRODUCTION
■
Dipeptidylpeptidase (DPP4) inhibitors,1 such as sitagliptin,2 sax-
agliptin,3 vildagliptin,4 and linagliptin,5 have emerged as a favor-
able class of agents constituting an effective therapeutic option in
the treatment of diabetes mellitus, a debilitating condition that
afflicts about 347 million people worldwide (WHO Fact Sheet
No. 312, Sep 2012). This is due in part to their ability to control
hyperglycemia alone or in combination with other agents and
with reduced risk of hypoglycemia and weight gain, side effects
that are common to several other oral antidiabetic therapies.
During our efforts to identify backup candidates to saxagliptin, a
number of novel chemotypes were discovered that less closely
resemble substrate mimetic scaffolds.6 Compounds that were
being evaluated in the clinic at the time had varied selectivities
versus DPP8 and DPP9, the significance of which was unknown.
A report7 from Merck on the potential liabilities of DPP8/9
inhibition using a DPP8/9-selective inhibitor in preclinical spe-
cies suggested that it may be desirable to maximize selectivity
versus DPP8 and DPP9. While a number of studies have subse-
quently suggested that inhibition of DPP8/9 is not associated
with adverse events in humans, these data were not published at
the time.7b−d We decided to include selectivity as a criterion in
our backup program to mitigate against any potential issues even
if the significance of DPP selectivity was unclear. Additionally, a
Received: June 17, 2013
© XXXX American Chemical Society
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Figure 1. Design of novel bicyclic cores.
and rapid functionalization in this solvent-exposed region for
optimization of activity and liability profiles.
converted into the carbamate via treatment with ClCO2Et/
Et3N/DMAP followed by deprotection with TFA to afford 1g.
The racemic intermediate 8b was resolved to homochiral 11a
and 11b using chiral supercritical fluid chromatography (SFC).
Following the sequence described in Scheme 1, a number of ho-
mochiral analogues 2 and 3, specified in Scheme 2, were syn-
thesized using resolved atropisomeric intermediates 11a and
11b. Stereochemistry of intermediates 11a and 11b was assigned
on the basis of inhibitory activities of pairs of enantiomers gen-
erated individually from 11a and 11b. The intermediate yielding
the more active antipode was assigned the stereochemistry
indicated in 11a. In addition, in every case in this and other
related bicyclic series for which we have X-ray cocrystal struc-
ture data (seven compounds from three different series 5-
oxopyrrolopyridines, 7-oxopyrrolopyridines, and imidazolopyr-
imidines), the more active atropisomer invariably had the Sa
stereochemistry.
Scheme 3 describes the general synthesis of amide-containing
5-oxopyrrolopyridines. Debenzylation of 9i by hydrogenolysis
afforded acid 15, which was subsequently reduced to the primary
alcohol 16 via acid chloride prepared in situ with a combination
of trichloroacetonitrile and triphenylphosphine10 followed by
treatment with lithium tri-tert-butoxyaluminum hydride.11 The
racemate 16 was resolved by chiral supercritical fluid chroma-
tography to give the desired atropisomer, which was converted to
the corresponding chloride 17 by treatment with methanesul-
fonyl chloride. The acid 18 was obtained in an uneventful three-
step sequence in 73% overall yield as shown in Scheme 3. Cou-
pling of 18 with appropriate amines followed by deprotection
yielded the target amides. The atropisomerically pure amino acid
pair of 2l and 3d was derived from 10h via conversion to the
primary amine, chiral chromatographic separation, and depro-
tection.
Our previous X-ray analyses of compounds from multiple
bicyclic series related to 1 had established that the more active
atropisomer in every case was Sa with the 2-Cl atom in 2 pointing
below the plane.6b−d On the basis of previous work on closely
related series in our group,6c,d the 2,4-dichlorophenyl group was
retained as an optimized aryl substitution at the 4-position of the
pyridine.
RESULTS AND DISCUSSION
■
Schemes 1−4 outline the synthesis of 5-oxopyrrolopyridines
with aryl, heteroaryl, and alkyl substituents on the lactam.
Dihydropyrrolopyridinones 1, 2, 3 could be prepared according
to the general synthesis outlined in Scheme 1. Knoevenagel con-
densation of ethyl chloroacetoacetate 4 with 2,4-dichloroben-
zaldehyde gave styrene 5. The dihydropyridyl ring was closed
by further condensation of styrene 5 with enamine 6 to yield
diester 7. Oxidation of the dihydro species to the fully aromatic
pyridine diester 8 was accomplished with nitric acid.9 Closure of
the pyrrolidone ring by treatment with primary amine under
microwave heating condition gave 5-oxopyrrolidinopyridine 9.
Debenzylation of 9b−g and 9s by hydrogenolysis or hydrolysis
of methyl ester 9a afforded the corresponding acid, which was
subsequently reduced to primary alcohol via anhydride formed
by treatment with ethyl chloroformate followed by reduction
with NaBH4. The alcohol was then converted into the corre-
sponding chloride 10 by treatment with methanesulfonyl
chloride. The desired primary amines 1, 2, and 3 were obtained
by reaction of the chloride with NH3/MeOH under microwave
heating conditions. Homochiral analogues 3e, 2a, and 3a were
isolated by using chiral supercritical fluid chromatography (SFC)
from the corresponding racemic precursors. The primary amine
1f was protected with Boc2O, and the resulting lactam was
An optimized, scalable protocol for the synthesis of 2s is
described in Scheme 4. Chloride 10s was converted to the
B
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a
demonstrated to be a significant issue, we also sought to mitigate
against any potential concerns associated with DPP8/9 inhi-
bition by maximizing DPP4 specificity. Ortho substitution on the
phenyl group (1c) resulted in a 5-fold increase in potency at
DPP4 and >1000-fold selectivity versus DPP8/9. X-ray structure
analysis of the analogous homochiral 7-oxodihydropyrrolopyr-
idinone (compound (+)-3f in ref 6c, PDB entry 3SX4) was
consistent with two conformations of the ortho-methoxy group.
In one orientation, the oxygen of the MeO group as well as the
carbonyl oxygen of the 7-oxo group showed electrostatic
interactions with Arg125. While an X-ray structure of 2a is not
available, one could speculate that a similar additional interac-
tion with the ortho-MeO group may have contributed to the
enhanced potency in the 5-oxo series as well. Essentially all
phenyl- and benzyl-substituted analogues in Table 1 showed
significant inhibition of CYP3A4. Introducing heteroaromatic
groups to reduce clogP helped reduce the CYP3A4 liability to
only a modest extent. For example, oxazole 2d, with the lowest
clogP in the table, showed an IC50 against CYP3A4 of 8 μM.
Alkyl-substituted analogues shown in Table 2, with the exception
of 2k, were relatively weaker inhibitors of CYP3A4. The hERG
liability was greatly minimized by alkyl substitution (1e, 2g, 3b).
Cyclic ethers (2h, 2i) as well as basic groups (2j) were well-
tolerated. However, the cyclic ethers were metabolically less
stable while the pyrrolidine 2j was about 10-fold less potent than
our desired goal. The most potent DPP4 inhibitor from this
series was 2k with a Ki of 0.4 nM and DPP8/9 selectivity of 9500/
7250, respectively. However, its IC50 vs CYP3A4 of 3 μM and
poor metabolic stability made it nonprogressible. A significant
finding was that the unsubstituted lactam 1f retained modest
DPP4 inhibitory activity and good DPP8/9 selectivity and had
minimal hERG and CYP3A4 liabilities (Table 3). The goal at this
point was to improve upon its DPP4 potency while retaining the
beneficial overall profile. Previous SAR findings in our group as
well as examination of several X-ray cocrystal structures of earlier
analogous compounds had suggested that the substituent on the
lactam nitrogen was exposed to solvent and hence an optimal
position to explore functionalities that would potentially retain
DPP4 activity and selectivity versus DPP8/9 and mitigate hERG
and CYP3A4 liabilities. We were pleased to find that homochiral
acids such as 2l and 2m and homochiral amides such as 2n, 2o,
and 2p, which lent themselves to facile SAR exploration,
exhibited potent DPP4 inhibition. While the acids showed
superior CYP3A4 profiles compared with the amides, they were
also generally more potent at DPP9 (for example, 2m, Ki =
300 nM). On the other hand, amides had Ki values of 7−10 μM
against CYP3A4. The most potent compound from Table 3, 2n,
suffered from poor metabolic stability in mouse liver micro-
somes. However, the finding triggered a concerted search, driven
by structure−activity and structure−liability data analysis, for an
optimal amide substituent with a superior profile. None of the
compounds in Table 4 displayed any hERG flux activity up to
80 μM. In general, lactam substituents containing a free acid,
nitrogen-containing basic groups, and primary and secondary
amide groups showed poor PAMPA permeability (data not
shown). PAMPA permeability at pH 7.4 for most neutral ali-
phatic and aromatic substituents as well as for tertiary amides was
high. Further optimization of the amide functionality (Table 4)
yielded compound 2s with an overall profile that was suitable
for progression into efficacy studies. Other close analogues that
were of comparable potency, selectivity, and CYP3A4 profile to
2s had inferior metabolic stability or poor permeability. For
example, 2r had a PAMPA permeability of 87 nm/s at pH 7.4
Scheme 1
a
Reagents and conditions: (a) 2,4-dichlorobenzaldehyde, BnNH2,
AcOH, IPA, 100%; (b) IPA, 79% 7a, 65% 7b; (c) AcOH, HNO3/H2O,
57% 8a, 68% 8b; (d) R2NH2, EtOH, microwave 150 °C, 71% 9a, 53%
9b, 71% 9s; (e) LiOH, THF, H2O, 19% for 9a or 10% Pd/C, H2,
EtOAc, 100% for 9b−s; (f) ClCO2Et, Et3N, THF, then NaBH4, THF,
H2O; (g) MsCl, Et3N, CH2Cl2, 33% two steps 10a, 7% two steps 10b,
66% two steps 10s; (h) 7 M NH3 in MeOH, microwave 100 °C, 79%
1b; (i) Boc2O, NaHCO3, THF; (j) ClCO2Et, Et3N, DMAP, CH2Cl2,
39% two steps; (k) TFA, CH2Cl2, 91% 1g; (l) chiral separation
(Chiralcel OJ 5 cm × 50 cm column; 20 μm; mobile phase, isocratic
gradient of 15% EtOH−MeOH (50%) in heptane); (m) supercritical
fluid chromatography (Chiralpak AD-H 250 mm × 30 mm i.d.; 5 μm;
flow rate, 120 mL/min; mobile phase, CO2/EtOH 75/25 with 0.1%
DEA).
bis-Boc-protected amino intermediate 19 by treatment with
KNBoc2. The racemate 19 was resolved using SFC to obtain the
desired atropisomeric intermediate 20, which after removal of
the Boc protecting groups under acidic conditions followed by
basification afforded 2s as a free base whose absolute stereo-
chemistry was confirmed by X-ray analysis (Figure 2). In general,
the stereochemistry of individual atropisomers was assigned on
the basis of X-ray cocrystal structures of about a dozen analogues
in the 5-oxo and multiple other bicyclic series.6b,c The more
active atropisomer was invariably significantly more potent than
the less active atropisomer (for example, see 2f vs 3c and 2g vs 3b
in Table 2 and 2l vs 3d in Table 3).
Analogous to our observations in the related 7-oxopyrrolopyr-
idine series,6c hERG, sodium channel, and CYP3A4 liabilities
were evident in the 5-oxopyrrolopyridine series when the R
group was aryl (Table 1). Mitigation of these liabilities was a
major goal of the program. Additionally, though not clearly
C
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a
Scheme 2
a
Reagents and conditions: (a) supercritical fluid chromatography (Chiralpak AD 250 mm × 4.6 mm i.d.; 10 μm; 35 °C; flow rate, 2.0 mL/min;
mobile phase, CO2/IPA 82/18; injection volume, 5 μL; detector wavelength, 220 nm); (b) R2NH2, EtOH, microwave 150 °C; (c) 10% Pd/C, H2,
EtOAc, 100%; (d) ClCO2Et, Et3N, THF, then NaBH4, THF, H2O; (e) MsCl, Et3N, CH2Cl2; (f) 7 M NH3 in MeOH, microwave 100 °C; (g) TFA,
CH2Cl2, 46%; (h) Boc2O, NaHCO3, THF, H2O; (i) Et2NH, PyBOP, DIEA, THF, 100% two steps.
versus 330 nm/s for 2s. Compound 2t, while more potent
and comparably selective versus DPP8/9, had a worse CYP3A4
and metabolic stability profile than 2s. The more active atro-
pisomer 2s, with the S stereochemistry (confirmed by X-ray
analysis, Figure 2), was 14-fold more potent than the less active
atropisomer 3e at inhibiting DPP4 and also showed a bigger
therapeutic window versus CYP3A4. Compound 2s had a DPP4
Ki of 0.9 nM and a DPP8/9 selectivity of 5400 and 3600,
respectively. The atropisomeric integrity was unchanged after
incubation of 2s in human and rat plasma for 3 h at 37 °C.
Compound 2s was further characterized by cocrystallization
within the active site of DPP4 (Figure 2).12a As with previous
X-ray structures resolved for earlier analogues within this che-
motype, the dominant nonhydrophobic interactions were
between the basic primary amine of 2s and the two glutamate
residues E206/E207 of DPP4. As was observed in the 7-oxo
series,6c the amide functionality on the lactam was exposed to
solvent. There were no interactions detected with the catalytic
Ser630. The amide carbonyl appeared to have a stacking
interaction with Tyr547, which may have contributed to DPP4
potency. Similar π−π interactions were observed in X-ray
structures of phenyl-substituted lactams as well. Overall, there
were fewer interactions in the S630-containing α/β-hydrolase
domain relative to saxagliptin,12b consistent with the hypothesis
that greater selectivity could be achieved by taking advantage
of a lower homology among DPP members in the β-propeller
domain.
The permeability (Pc) in the PAMPA model was pH-
dependent and, at pH ≥ 6.5 (typical pH range in human intes-
tine), was similar to Pc values of compounds that exhibit good
(>80%) absorption in humans (Table 5). The basolateral-to-apical
permeability of 2s was ≥5-fold higher versus apical-to-basolateral
permeability in Caco-2 cells (pH ≥ 6.5; data shown are at pH
7.4), suggesting that it is a substrate of efflux transporters. The
percent of 2s bound to serum proteins was low in all species
tested. The rate of 2s metabolism in vitro (liver microsomes and
hepatocytes) was low to moderate, and a low hepatic clearance in
humans was predicted. In vivo, the total blood clearance of 2s was
low to intermediate in rats, dogs, and monkeys but high in mice
(Table 6). Overall, the elimination of 2s appeared to be mediated
by both hepatic metabolism and renal excretion, with a minor
contribution from biliary excretion. Compound 2s had a
favorable pharmacokinetic profile and good absorption proper-
ties with an absolute oral bioavailability ranging from 41% to 95%
across animal species.
Oral Glucose Tolerance in ob/ob Mice. On the basis of its
in vitro potency, selectivity, PK, and a favorable profile in in vitro
liability assays, compound 2s was selected for detailed evaluation
D
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a
mouse PK study, Cmax and Tmax for 2s were determined to be
56 nM and 1 h after an oral dose of 0.82 mpk, respectively. With a
mouse plasma free fraction of 14%, coverage over DPP4 Ki would
be expected during the efficacy study.
Scheme 3
These effects on insulin translated into decreases in peak glucose
concentrations (Figure 4). All doses of 2s above 0.3 μmol/kg
gave maximal decreases in peak glucose, similar to that seen
with the 3 μmol/kg dose of BMS-538305.13
The selection of 2s as the clinical candidate was arrived at after
an extensive analysis of all available in vitro and in vivo activity
and liability data for advanced leads from multiple distinct che-
motypes. In particular, differentiating areas among advanced
leads pertained to metabolism, reactive intermediate formation,
pharmacokinetic properties, and overall target potency. A desir-
able profile of the candidate included having a DPP4 potency
comparable to that of saxagliptin (∼1 nM) and in vitro selectivity
ratios versus DPP8/9 that were significantly higher than those for
saxagliptin. Although the 7-oxopyrrolidinone series described
earlier6c was the most potent series with Ki values for leads in
the 300−600 pM range, the series suffered from the poorest
pharmacokinetic properties and was therefore not advanced. In
the imidazopyrimidine6a,b and the 5-oxopyrrolidinone series, a
number of compounds showed evidence of mild time-dependent
CYP3A4 inhibition. After analysis of over 100 analogues in this
assay, candidates were advanced on the basis of a lack of time
dependency and weakest overall inhibition potency. Addition-
ally, several analogues showed evidence of reactive intermediate
formation in glutathione trapping studies, presumably through
the intermediacy of an oxime group arising from the benzylamine
functionality. Only compounds that showed acceptable profiles
in this reactive intermediate assays were advanced further.
Further detailed comparative analysis of the full profiles of the
best leads from each series resulted in the selection of 2s as a
candidate for toxicological evaluation in cynomolgous monkeys,
where 2s was determined to be acceptable for advancement
to the clinic.
a
Reagents and conditions: (a) H2, 10% Pd/C, 100%; (b) CCl3CN,
Ph3P resin, CH2Cl2; (c) LiAl(OBu)3H, THF, 0 °C, 80% two steps; (d)
supercritical fluid chromatography (Chiralpak AD 250 mm × 4.6 mm
i.d.; 10 μm; 35 °C; flow rate, 2.0 mL/min; mobile phase, CO2/IPA
82/18; injection volume, 5 μL; detector wavelength, 220 nm); (e)
MsCl, Et3N, CH2Cl2, 0 °C, >90%; (f) 7 M NH3 in MeOH, 50 °C; (g)
TFA, CH2Cl2; (h) Boc2O, NaHCO3, THF, H2O, 73% three steps; (i)
HOBt·H2O, EDCI, NH4Cl, DIEA, DMF, 2q; amine, PyBOP, DIEA,
THF, 2n, 2r, 2s, 2t, 2u, 2v; (j) TFA, CH2Cl2, 12−74% two steps.
a
Scheme 4
The toxicology profile of 2s was evaluated in a battery of in
vitro safety tests as well as in vivo tests in rats and dogs. No
adverse effects were identified after single (up to 500 mg/kg) or
repeated (up to 300 mg/kg) oral dosing for up to 14 days in rats.
No clinical chemistry or hemotology changes were seen with 2s.
In the dog, administration of 2s once daily for 5 days produced
no significant dose-related changes at doses up to 25 mg/kg. At
50 mg/kg, a softening of stools was noted. Compound 2s did not
demonstrate any potential for undesirable cardiovascular activity
at an oral dose of 50 mg/kg. On the basis of the results of human
in vitro assays and allometrically scaled animal data, 2s was pre-
dicted to be a low clearance compound in human with a pro-
jected human efficacious dose of 13−33 mg q.d. Because of the
absence of any significant dose-limiting toxicities in preclinical
toxicology studies, 2s was considered to have demonstrated an
overall efficacy, safety, and developability profile suitable for
advancement to the clinic, allowing for exploring a hypothesis
that higher sustained exposures above what are required for
maximal DPP4 inhibition of a potent and selective DPP4 inhib-
itor with an acceptable safety margin would achieve greater
HbA1c reductions in humans.
a
Reagents and conditions: (a) KNBoc2, THF/DMF (15:5), 60 °C,
60%; (b) supercritical fluid chromatography (Chiralpak AD 250 mm ×
4.6 mm i.d.; 10 μm; 35 °C; flow rate, 2.0 mL/min; mobile phase, CO2/
IPA 82/18; injection volume, 5 μL; detector wavelength, 220 nm); (c)
4 N HCl, dioxane; (d) 1 N NaOH, CH2Cl2, 86% two steps.
in in vivo models of efficacy and safety. When tested in vivo in
insulin resistant high fat fed (HFD) ob/ob mice, which were
given an oral glucose tolerance test (oGTT) 1 or 4 h after dose,
2s demonstrated an insulinotropic effect at all doses, with insulin
AUCs increasing 2-, 3.8-, 2.5-, and 3.1-fold over the vehicle
response at the 0.3, 1, 3, and 10 μmol/kg dose levels, respectively
(Figure 3). The 0.3 and 3 μmol/kg dose treatment groups did
not achieve statistical significance in this study. In a separate
EXPERIMENTAL SECTION
■
All reactions were carried out under a static atmosphere of argon or
nitrogen, and mixtures were stirred magnetically unless otherwise noted.
All reagents used were of commercial quality and were obtained from
Aldrich Chemical Co., Sigma Chemical Co., Lancaster Chemical Co.,
E
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Figure 2. X-ray cocrystal structure of 2s bound to the DPP4 active site at 2.35 Å resolution.
Table 1. N-Aryl- and N-Benzyl-5-oxopyrrolopyridines
a
b
c
d
n = 1−3. Versus substrate 7-benzyloxy-4-trifluoromethyl coumarin. ND = not determined. Incubation in mouse liver microsomes for 10 min at
3 μM compound and 1 mg/mL protein.
1
or Acros Chemical Co. 1H (400 MHz) and 13C (100 MHz) NMR spec-
tra were recorded on a JEOL GSX400 spectrometer using Me4Si as
an internal standard unless otherwise noted. H (500 MHz) and 13C
(125 MHz) NMR spectra were recorded on a JEOL JNM-ECP500
F
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Table 2. N-Alkyl-5-oxopyrrolopyridines
a
b
c
d
n = 1−3. Versus substrate 7-benzyloxy-4-trifluoromethyl coumarin. ND = not determined. Incubation in mouse liver microsomes for 10 min at
3 μM compound and 1 mg/mL protein.
Table 3. Acid-, Ester-, and Amide-Containing 5-Oxopyrrolopyridines
a
b
c
d
n = 1−3. Versus substrate 7-benzyloxy-4-trifluoromethyl coumarin. ND = not determined. Incubation in mouse liver microsomes for 10 min at
3 μM compound and 1 mg/mL protein.
spectrometer. Chemical shifts are given in parts per million (ppm)
downfield from internal reference Me4Si in δ units, and coupling
constants (J) are given in hertz (Hz). Selected data are reported in the
following manner: chemical shift, multiplicity, coupling constants. All
reactions were carried out using commercially available anhydrous
solvents from Aldrich Chemical Co. or EM Science Chemical Co. unless
otherwise noted. All flash chromatographic separations were performed
using E. Merck silica gel (particle size, 0.040−0.063 mm). Reactions
were monitored by TLC using 0.25 mm E. Merck silica gel plates
(60 F254) and were visualized with UV light, with 5% phosphomolybdic
G
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Table 4. Acetamide-Substituted 5-Oxopyrrolopyridines
a
b
c
n = 1−3, except for 2s (n = 32). Versus substrate 7-benzyloxy-4-trifluoromethylcoumarin. Incubation in mouse liver microsomes for 10 min at
3 μM compound and 1 mg/mL protein.
were removed by rotary evaporation under vacuum using a standard
rotovap equipped with a dry ice condenser. All filtrations were per-
formed with a vacuum unless otherwise noted. All final compounds were
purified to >95% purity by HPLC analysis.
Table 5. Permeability and Plasma Protein Binding Profile
of 2s
Pc, nm/s (PAMPA) (n = 3)
(Z)-Ethyl 4-Chloro-2-(2,4-dichlorobenzylidene)-3-oxobuta-
noate (5). A solution 2,4-dichlorobenzaldehyde (4.6 g, 26.1 mmol),
ethyl 4-chloro-3-oxobutanoate (4.5 g, 27.4 mmol), benzylamine (165 mg,
1.5 mmol), and acetic acid (118 mg, 2.0 mmol) in isopropyl alcohol
(30 mL) was stirred at ambient temperature for 96 h. The mixture was
diluted with isopropyl alcohol to give a total volume of 50 mL and was
saved as a stock solution (0.52 mmol/mL).
pH 5.5
pH 6.5
pH 7.4
19.7 3.8
129.7 12.9
330 49
a
Pc, nm/s (Caco-2)
apical to basolateral
basolateral to apical
26
1
131 18
(E)-Benzyl 3-Aminobut-2-enoate (6b). A mixture of benzyl aceto-
acetate 4.6 g, 24 mmol) and ammonium acetate (9.2 g, 119.5 mmol) in
methanol (30 mL) was allowed to stir at ambient temperature for 72 h.
The solvent was evaporated, and the residue was taken up in CHCl3/
H2O. The combined organic layer was washed with brine, dried
(Na2SO4), and evaporated to give 6b (4.3 g, 90% yield) as a golden oil.
1H NMR (400 MHz, CDCl3) δ 91 (s, 3H), 4.60 (s, 1H), 5.12 (s, 2H),
plasma protein binding, % free
human
mouse
rat
18
14
28
27
16
dog
monkey
a
7.24−7.40 (m, 5H).
Measured at pH 7.4.
Ethyl 2-(Chloromethyl)-4-(2,4-dichlorophenyl)-5-(2-me-
thoxy-2-oxoethyl)-6-methyl-1,4-dihydropyridine-3-carboxy-
late (7a). A mixture of 2,4-dichlorobenzaldehyde (2.3 g, 12.9 mmol),
ethyl 4-chloro-3-oxobutanoate (2.2 g, 12.9 mmol), benzylamine (80.0 mg,
0.8 mmol), and acetic acid (55.0 mg, 0.9 mmol) in isopropyl alcohol
(15 mL) was stirred at ambient temperature for 65 h. 6a (1.6 g,
14.4 mmol) was added to the reaction mixture, and stirring continued at
ambient temperature for 24 h. The reaction was quenched with con-
centrated HCl (1 mL), and the mixture was stirred at ambient tem-
perature for 2 h. The reaction mixture was then concentrated in vacuo,
diluted with diethyl ether, filtered, and evaporated. The residue was
purified by flash chromatography (120 g column, 0−100% EtOAc/
hexanes) to yield 7a (4.2 g, 79% yield) as a yellow sticky oil. 1H NMR
(400 MHz, CDCl3) δ 1.22 (t, J = 7.5 Hz, 3H), 2.36 (s, 3H), 3.63 (s, 3H),
4.11 (q, J = 7.2 Hz, 2H), 4.86 and 4.97 (ABq, J = 14.0 Hz, 2H), 5.40 (s,
1H), 6.40 (broad s, 1H), 7.13 (dd, J = 8.4, 2.2 Hz, 1H), 7.26−7.31 (m,
2H). m/z = 418 [M + H]+.
3-Benzyl 5-Ethyl 6-(chloromethyl)-4-(2,4-dichlorophenyl)-2-
methyl-1,4-dihydropyridine-3,5-dicarboxylate (7b). A mixture of
stock solution 5 (25 mL, 13 mmol) and 6b (2.8 g, 14.5 mmol) in
isopropyl alcohol (3 mL) was allowed to stir at ambient temperature
for 18 h. The reaction was quenched with concentrated HCl (8 mL),
and the mixture was stirred at ambient temperature for 2 h. The mi-
xture was concentrated in vacuo, diluted with diethyl ether, filtered, and
a
Table 6. Pharmacokinetic Profile of 2s
mouse
rat
dog
monkey
iv dose (mg/kg)
po dose (mg/kg)
CL (mL min−1 kg−1
0.82
0.82
137
6.3
0.82
0.82
40.9
2.9
3
3
3
3
)
10.6
2.7
7.8
95
19.2
2.8
5.6
84
V
ss (L/kg)
1/2 (h)
T
1.8
1.5
bioavailability (%)
41
48
a
Vehicle used in iv and po studies in all species: water.
acid in 95% EtOH or by a sequential treatment with 1 N HCl/MeOH
followed by ninhydrin staining. LC/MS data were recorded on a
Shimadzu LC-10AT equipped with a SIL-10A injector, a SPD-10AV
detector normally operating at 220 nm, and interfaced with a Micromass
ZMD mass spectrometer. HPLC retention times, unless otherwise
noted, are reported using a Zorbax SB C18 4.6 mm × 75 mm column.
The gradient solvent system was from 100% A/0% B to 0% A/100% B
(A = 90% H2O/10% MeOH + 0.2% H3PO4) (B = 90% MeOH/10%
H2O + 0.2% H3PO4) for 8 min. Detection was at 220 nm. All solvents
H
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Figure 3. Insulin response and effects on plasma insulin AUC during an oGTT in an ob/ob mouse model, following treatment with 2s.
Figure 4. Glucose response and effects on glucose AUC during an oGTT in an ob/ob mouse model, following treatment with 2s.
25
evaporated. The residue was purified by flash chromatography (120 g
column, EtOAc/hexanes) to give 7b (4.2 g, 65% yield) as a yellow sticky
oil. 1H NMR (400 MHz, CDCl3) δ 1.19 (t, J = 7.0 Hz, 3H), 2.35 (s, 3H),
4.05−4.15 (m, 2H), 4.82 and 4.97 (ABq, J = 14.1 Hz, 2H), 5.07 and 5.11
(ABq, J = 12.3 Hz, 2H), 5.41 (s, 1H), 6.37 (broad s, 1H), 7.07 (dd, J =
8.4, 2.2 Hz, 1H), 7.16−7.32 (m, 5H), 7.35−7.38 (m, 2H).
3-Ethyl 5-Methyl 2-(chloromethyl)-4-(2,4-dichlorophenyl)-6-
methylpyridine-3,5-dicarboxylate (8a). 7a (3.4 g, 8.1 mmol) was
dissolved in acetic acid (15 mL) and 70% nitric acid/water (15 mL). The
reaction mixture was stirred at ambient temperature for 72 h. The crude
product (3.7 g) was purified by flash chromatography (120 g column,
0−100% EtOAc/hexanes) to give 8a (1.9 g, 57% yield) as a pale yellow
oil. 1H NMR (400 MHz, CDCl3) δ 1.02 (t, J = 7.7 Hz, 3H), 2.66 (s, 3H),
3.60 (s, 3H), 4.10 (q, J = 7.0 Hz, 2H), 4.80 and 4.93 (ABq, J = 11.0 Hz,
2H), 7.13 (d, J = 7.0 Hz, 1H), 7.26−7.33 (m, 1H), 7.45 (s, 1H). m/z =
416 [M + H]+.
3-Benzyl 5-Ethyl 6-(chloromethyl)-4-(2,4-dichlorophenyl)-2-
methylpyridine-3,5-dicarboxylate (8b). 7b (4.1 mg, 8.2 mmol) was
dissolved in acetic acid (30 mL) and 70% nitric acid/water (25 mL). The
reaction mixture was allowed to stir at ambient temperature for 18 h.
The crude product (4.2 g) was purified by flash chromatography (120 g
column, 0−100% EtOAc/hexanes) to give 8b (2.7 g, 68% yield) as a pale
yellow oil. 1H NMR (400 MHz, CDCl3) δ 0.98 (t, J = 7.3 Hz, 3H), 2.65
(s, 3H), 4.05 (q, J = 7.0 Hz, 2H), 4.77 and 4.92 (ABq, J = 11.0 Hz, 2H),
5.04 (s, 2H), 7.01 (d, J = 8.35 Hz, 1H), 7.07 (dd, J = 8.4, 2.2 Hz, 1H),
7.10−7.14 (m, 2H), 7.21 (d, J = 2.2 Hz, 1H), 7.28−7.28 (m, 3H). m/z =
492 [M + H]+.
(S)-3-Benzyl 5-Ethyl 6-(chloromethyl)-4-(2,4-dichlorophen-
yl)-2-methylpyridine-3,5-dicarboxylate (11a) and (R)-3-Benzyl
5-Ethyl 6-(chloromethyl)-4-(2,4-dichlorophenyl)-2-methylpyri-
dine-3,5-dicarboxylate (11b). 8b was separated into individual atro-
pisomers using supercritical fluid chromatography (SFC). Conditions:
Whelk O-1 SS, 500 mm × 20 mm, 10 μm; 35 °C; 5% IPA with 0.1% DEA
in SFC−CO2; 100 bar; 60 mL/min; 220 nm. Faster-eluting isomer: 11a.
[α]D +54.45° (c 10.21 mg/mL, CHCl3). Analytical HPLC: gradient
solvent system from 100% A/0% B to 0% A/100% B (A = 90% H2O/
10%MeOH + 0.2% H3PO4) (B = 90% MeOH/10% H2O + 0.2% H3PO4)
for 4 min; detection at 220 nm. YMC S5 ODS Ballistic 4.6 mm × 50 mm
column; tR = 4.8 min. Chiral analytical HPLC: tR = 7.39 min on analytical
SFC [Regis (S,S) Whelk-O1 250 mm × 4.6 mm i.d.; 10 μm (S/N
200036); mobile phase CO2/IPA, 95/5; flow rate, 2.0 mL/min]. Slower-
eluting isomer: 11b. [α]D25 −52.87° (c 1.021, CHCl3). tR = 7.92 min on
chiral analytical SFC.
Methyl 4-(2,4-Dichlorophenyl)-2,7-dimethyl-5-oxo-6-phe-
nyl-6,7-dihydro-5H-pyrrolo[3,4-b]pyridine-3-carboxylate (9a).
A mixture of 8a (671 mg, 1.6 mmol) and aniline (168 mg, 1.8 mmol)
in ethanol (5 mL) was heated to 175 °C for 45 min in a microwave
reactor. The mixture was purified by flash chromatography (120 g
column, 0−100% EtOAc/hexanes) to give 9a (485 mg, 71% yield) as a
pale brown solid. 1H NMR (400 MHz, CDCl3) δ 2.77 (s, 3H), 3.64 (s,
3H), 4.88 and 5.00 (ABq, J = 17.6 Hz, 2H), 7.13−7.20 (m, 2H), 7.33 (dd,
J = 8.4, 2.2 Hz, 1H), 7.36−7.44 (m, 2H), 7.53 (d, J = 1.8 Hz, 1H), 7.79
(d, J = 7.9 Hz, 2H). m/z = 473 [M + H]+.
Benzyl 4-(2,4-Dichlorophenyl)-6-(4-methoxyphenyl)-2-
methyl-5-oxo-6,7-dihydro-5H-pyrrolo[3,4-b]pyridine-3-car-
boxylate (9b). A mixture of 8b (199 mg, 0.4 mmol) and 4-
methoxybenzenamine (61 mg, 0.5 mmol) in ethanol (3 mL) was heat-
ed in a microwave reactor at 175 °C for 15 min. The solvent was
evaporated and the residue was purified by flash chromatography (12 g
column, EtOAc/hexanes) to give 9b (114.6 mg, 53% yield) as a golden
oil. 1H NMR (400 MHz, CDCl3) δ 2.76 (s, 3H), 3.79 (s, 3H), 4.81 and
4.90 (ABq, J = 17.6 Hz, 2H), 5.05 and 5.11 (ABq, J = 11.9 Hz, 2H), 6.89
(d, J = 9.2 Hz, 2H), 7.03 (d, J = 8.4 Hz, 1H), 7.08−7.16 (m, 3H), 7.28−
7.38 (m, 4H), 7.66 (d, J = 9.2 Hz, 2H). m/z = 533 [M + H]+.
Benzyl 4-(2,4-Dichlorophenyl)-6-(2-(dimethylamino)-2-ox-
oethyl)-2-methyl-5-oxo-6,7-dihydro-5H-pyrrolo[3,4-b]-
pyridine-3-carboxylate (9s). 8b (72.0 g, 0.146 mol) was dissolved in
dimethylacetamide (130 mL). This solution was added in three portions
I
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as described below. To a mixture of glycine dimethylamide hydro-
chloride salt (25.9 g, 0.187 mol) in dimethylacetamide (150 mL) at
room temperature was added triethylamine (122.5 mL, 0.879 mol). The
resulting slurry was heated at 80 °C. The solution of chloride 8b in DMA
(60 mL) as previously described was added over a period of 10 min. The
resultant mixture was stirred for an additional 50 min period. A second
portion of the chloride solution (60 mL) was added over a period of
10 min. The mixture was then stirred at this temperature for 1.5 h. The
remainder of the chloride solution was added over a period of 10 min,
and the resulting mixture was stirred for an additional 2 h. After the
mixture was cooled to room temperature, low boiling volatiles were
removed under reduced pressure. The mixture was diluted with H2O
(3 L) and allowed to stand at room temperature overnight. The liquid
layer was decanted, and the solid residue was dissolved in EtOAc (3 L)
(required heating at 50 °C with stirring). The organic layer was washed
with H2O (130 mL), 0.5 N HCl (30 mL), and H2O (50 mL) and dried
(MgSO4). The organic layer was concentrated to approximately 250 mL
total volume and subjected to filtration to give the desired product
(48.17 g) as a beige solid. The mother liquor was concentrated and
subjected to recrystallization (EtOAc) to give a second portion of the
desired product (5.27 g, 71% overall yield). 1H NMR (400 MHz,
CDCl3) δ 2.73 (s, 3H), 2.93 (s, 3H), 3.01 (s, 3H), 4.23 and 4.48 (ABq,
JAB = 16.7 Hz, 2H), 4.62 and 4.70 (ABq, JAB = 17.5 Hz, 2H), 5.04 and
5.09 (ABq, JAB = 11.8 Hz, 2H), 7.00 (d, J = 8.3 Hz, 1H), 7.05−7.15 (m,
3H), 7.20−7.38 (m, 4H). 13C NMR (125 MHz, CDCl3) δ 172.28,
167.17, 166.68, 165.77, 163.36, 160.21, 142.44, 135.28, 134.37, 133.76,
131.07, 130.24, 129.00, 128.89, 128.58, 128.46, 126.54, 120.95, 67.66,
52.05, 43.27, 36.38, 35.63, 23.66. HPLC (gradient from 100% A/0% B to
0% A/100% B (A = 90% H2O/10% MeOH + 0.2% H3PO4) (B = 90%
MeOH/10% H2O + 0.2% H3PO4) for 8 min; detection at 220 nm.
Zorbax SB C18 4.6 mm × 75 mm column): tR = 7.97 min. m/z = 512
[M + H]+.
3-(Chloromethyl)-4-(2,4-dichlorophenyl)-2,7-dimethyl-6-
phenyl-6,7-dihydropyrrolo[3,4-b]pyridin-5-one (10a). A mixture
of 9a (183 mg, 0.43 mmol) and lithium hydroxide (∼200 mg) in THF/
H2O (4 mL) was allowed to stir at ambient temperature for 18 h. The
mixture was then heated in a microwave reactor for 1 h at 120 °C. The
reaction was quenched with 1 N HCl, and the aqueous portion was
extracted into EtOAc. The combined organic layer was washed with
brine, dried (Na2SO4), and evaporated to give the crude acid product
(34 mg, 19% yield) as a pale yellow solid. 1H NMR (400 MHz, CDCl3) δ
2.80 (s, 3H), 4.85 and 4.95 (ABq, J = 17.6 Hz, 2H), 7.15 (t, J = 7.5 Hz,
1H), 7.20 (d, J = 7.9 Hz, 1H), 7.30 (dd, J = 8.9, 1.8 Hz, 1H), 7.37 (t, J =
7.9 Hz, 2H), 7.49 (d, J = 2.2 Hz, 1H), 7.76 (d, J = 7.9 Hz, 2H). m/z = 413
[M + H]+. To a solution of the acid (25 mg, 0.06 mmol) in THF was
added ethyl chloroformate (24 μL, 0.25 mmol) followed by addition of
triethylamine (50 μL, 0.36 mmol). Immediate precipitation was seen.
The mixture was stirred at ambient temperature for 2 h, filtered, and
washed with THF (1 mL × 2). NaBH4 (11 mg, 0.29 mmol) in H2O
(0.3 mL) was added dropwise to the filtrate. The mixture was allowed to
stir at ambient temperature for 18 h and then diluted with EtOAc/H2O.
The organic layer was washed with brine, dried (Na2SO4), and evap-
orated to give the alcohol (11.5 mg) as a colorless oil. To a solution of
the alcohol (11.5 mg, 0.03 mmol) and triethylamine (30 μL, 0.22 mmol)
in dichloromethane (2 mL) wasadded mesyl chloride (15 μL, 0.19 mmol).
The mixture was allowed to stir at ambient temperature for 4 h.
The solvent was evaporated, and the residue was purified by flash chro-
matography (4 g column, 0−100% EtOAc/hexanes) to give 10a (4 mg,
33%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 2.89 (s, 3H), 4.31
and 4.56 (ABq, J = 11.9 Hz, 2H), 4.87 and 4.93 (ABq, J = 17.6 Hz, 2H),
7.16 (t, J = 7.5 Hz, 1H), 7.29 (d, J = 8.4 Hz, 1H), 7.38 (t, J = 7.9 Hz, 2H),
7.42 (dd, J = 8.1, 2.0 Hz, 1H), 7.57 (d, J = 2.2 Hz, 1H), 7.78 (d, J =
7.9 Hz, 2H). m/z = 418 [M + H]+.
triethylamine (150 μL, 1.1 mmol) were added sequentially. The mixture
was stirred at ambient temperature for 2 h and then filtered. NaBH4
(50 mg, 1.3 mmol) was added to the filtrate and the mixture stirred at
ambient temperature for 18 h. The reaction was quenched with satu-
rated aqueous NaHCO3 and extracted into EtOAc. The combined
organic layer was washed with brine, dried (Na2SO4), and evaporated.
The residue was purified by flash chromatography (12 g column,
EtOAc/hexanes) to give the alcohol (10.5 mg, 10% yield) as a white
solid. m/z = 429 [M + H]+. To a solution of the alcohol (10 mg,
0.02 mmol) in dichloromethane (4 mL) were added triethylamine
(25 μL, 0.18 mmol) and mesyl chloride (20 μL, 0.26 mmol). The mix-
ture was allowed to stir at ambient temperature for 18 h and was
evaporated. The residue was purified by flash chromatography (4 g
column, EtOAc/hexanes) to give 10b (7 mg, 67% yield) as a colorless
oil. 1H NMR (400 MHz, CDCl3) δ 2.89 (s, 3H), 3.80 (s, 3H), 4.31 and
4.56 (ABq, J = 11.9 Hz, 2H), 4.82 and 4.89 (ABq, J = 17.1 Hz, 2H), 6.87−
6.94 (m, 2H), 7.29 (d, J = 7.9 Hz, 1H), 7.41 (dd, J = 7.9, 1.8 Hz, 1H),
7.56 (d, J = 1.8 Hz, 1H), 7.63−7.70 (m, 2H). m/z = 447 [M + H]+.
2-(3-(Chloromethyl)-4-(2,4-dichlorophenyl)-2-methyl-5-oxo-
5H-pyrrolo[3,4-b]pyridin-6(7H)-yl)-N,N-dimethylacetamide
(10s). 1H NMR (400 MHz, CDCl3) δ 2.85 (s, 3H), 2.92 (s, 3H), 3.00 (s,
3H), 4.23 and 4.47 (ABq, JAB = 16.3 Hz, 2H), 4.29 and 4.54 (ABq, JAB
=
11.9 Hz, 2H), 4.60 and 4.69 (ABq, JAB = 17.5 Hz, 2H), 7.25 (d, J =
8.3 Hz, 1H), 7.37 (dd, J = 8.3, 2.2 Hz, 1H), 7.52 (d, J = 2.2 Hz, 1H). 13C
NMR (125 MHz, CDCl3) δ 67.22, 166.17, 162.90, 162.62, 143.95,
135.58, 133.60, 130.99, 130.75, 129.27, 127.09, 121.57, 51.95, 43.29,
39.83, 36.35, 35.63, 22.99. HPLC (gradient from 100% A/0% B to 0%
A/100% B (A = 90% H2O/10% MeOH + 0.2% H3PO4) (B = 90%
MeOH/10% H2O + 0.2% H3PO4) for 4 min; detection at 220 nm. YMC
S5 ODS 4.6 mm × 50 mm, Ballistic column): tR = 3.81 min. m/z = 427
[M + H]+.
3-(Aminomethyl)-4-(2,4-dichlorophenyl)-6-(4-methoxy-
phenyl)-2-methyl-6,7-dihydropyrrolo[3,4-b]pyridin-5-one, TFA
Salt (1b). A mixture of 10b (7 mg, 0.02 mmol) and 7 M NH3 in MeOH
(4 mL) was heated in a microwave reactor at 100 °C for 15 min. The
solvent was removed in vacuo, and the residue was purified by pre-
parative HPLC (Phenomenex, 10 min gradient, 30−100% B) to give 1b
(6.7 mg, 79% yield) as a TFA salt. 1H NMR (400 MHz, CDCl3) δ 2.86
(s, 3H), 3.80 (s, 3H), 3.97 and 4.20 (ABq, J = 14.5 Hz, 2H), 4.96 and 5.01
(ABq, J = 18.0 Hz, 2H), 6.94−7.00 (m, 2H), 7.36 (d, J = 8.4 Hz, 1H),
7.54 (dd, J = 8.4, 2.2 Hz, 1H), 7.59−7.65 (m, 2H), 7.72 (d, J = 1.8 Hz,
1H). HRMS calculated for C22H20Cl2N3O2: 428.0933. Found: 428.0943.
Analytical HPLC method: tR = 6.1 min.
3-(Aminomethyl)-4-(2,4-dichlorophenyl)-2-methyl-6-phe-
nyl-6,7-dihydropyrrolo[3,4-b]pyridin-5-one, TFA Salt (1a). 1H
NMR (400 MHz, CDCl3 + CD3OD as cosolvent) δ 2.83 (s, 3H), 3.88
and 4.10 (ABq, J = 14.5 Hz, 2H), 4.85 and 4.90 (ABq, J = 18.5 Hz, 2H),
7.14 (t, J = 7.0 Hz, 1H), 7.24−7.27 (m, 1H), 7.34 (t, J = 7.9 Hz, 2H), 7.40
(dd, J = 8.1, 2.0 Hz, 1H), 7.55 (d, J = 1.8 Hz, 1H), 7.70 (d, J = 8.8 Hz,
2H). HRMS calculated for C21H18Cl2N3O: 398.0827. Found: 398.0812.
3-(Aminomethyl)-4-(2,4-dichlorophenyl)-6-(2-methoxy-
phenyl)-2-methyl-6,7-dihydropyrrolo[3,4-b]pyridin-5-one, TFA
1
Salt (1c). H NMR (400 MHz, CDCl3) δ 2.87 (s, 3H), 3.83 (s, 3H),
3.99 and 4.22 (ABq, J = 14.5 Hz, 2H), 4.85−4.95 (m, 2H), 7.01 (broad t,
J = 7.5 Hz, 1H), 7.14 (broad d, J = 8.4 Hz, 1H), 7.32 (dd, J = 7.9, 1.8 Hz,
1H), 7.34−7.40 (m, 2H), 7.52 (dd, J = 8.4, 2.2 Hz, 1H), 7.69 (d, J =
2.2 Hz, 1H). HRMS calculated for C22H20Cl2N3O2: 428.0933. Found:
428.0937.
3-(Aminomethyl)-4-((S)-2,4-dichlorophenyl)-6-(2-methoxy-
phenyl)-2-methyl-6,7-dihydropyrrolo[3,4-b]pyridin-5-one, TFA
Salt (2a) and 3-(Aminomethyl)-4-((R)-2,4-dichlorophenyl)-6-(2-
methoxyphenyl)-2-methyl-6,7-dihydropyrrolo[3,4-b]pyridin-5-
one, TFA Salt (3a). 1c was separated on a Chiralcel OJ, 20 μm, 5 cm ×
50 cm column using an isocratic gradient of 15% EtOH−MeOH (50%)
in heptane to afford individual enantiomers 2a and 3a. 2a (faster-eluting):
[α]D25 +8.2°. Purity by chiral analytical HPLC [Chiralcel OJ 4.6 mm ×
250 mm; 15% EtOH−MeOH (1:1; containing 0.1% DEA) in heptane
3-(Chloromethyl)-4-(2,4-dichlorophenyl)-6-(4-methoxy-
phenyl)-2-methyl-6,7-dihydropyrrolo[3,4-b]pyridin-5-one
(10b). A mixture of 9b (114.6 mg, 0.2 mmol) and 10% Pd/C (44 mg) in
ethyl acetate (15 mL) was stirred under a H2 (g) balloon at ambient
temperature for 5 h. The reaction mixture was filtered and the filtrate
evaporated to give the acid (114 mg, 0.3 mmol). The acid was dis-
solved in THF (10 mL), and ethyl chloroformate (50 μL, 0.5 mmol) and
25
(containing 0.1% DEA)]: >98%. 3a (slower-eluting): [α]D −8.0°.
Purity by chiral analytical HPLC [Chiralcel OJ 4.6 mm × 250 mm; 15%
EtOH−MeOH (1:1; containing 0.1% DEA) in heptane (containing
0.1% DEA)]: >98%. The more potent atropisomer, 3a, was assigned the
J
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Sa stereochemistry based on analogy to a cocrystal structure of a close
analogue (compound (+)-3f in ref 6c). Analytical HPLC: tR = 4.8 min.
3-(Aminomethyl)-4-(2,4-dichlorophenyl)-6-(2-methoxyeth-
yl)-2-methyl-6,7-dihydropyrrolo[3,4-b]pyridin-5-one, TFA Salt
(1e, BMS-744891). 1H NMR (400 MHz, CDCl3) δ 2.83 (s, 3H), 3.34
(s, 3H), 3.58−3.63 (m, 2H), 3.65−3.78 (m, 2H), 3.95 and 4.18 (ABq, J =
14.5 Hz, 2H), 4.62 (s, 2H), 7.33 (d, J = 8.4 Hz, 1H), 7.52 (dd, J = 8.1,
2.0 Hz, 1H), 7.70 (d, J = 2.2 Hz, 1H). HRMS calculated for C18H20Cl2N3O2:
380.0933. Found: 380.0941. An X-ray cocrystal structure of 1e/DPP4
was obtained.12c It is presumed that the more active atropisomer (2f)
crystallized with the protein under the crystallization conditions.
3-(Aminomethyl)-4-(2,4-dichlorophenyl)-2-methyl-6,7-
dihydropyrrolo[3,4-b]pyridin-5-one, TFA Salt (1f). 1H NMR
(400 MHz, CDCl3) δ 2.84 (s, 3H), 3.95 and 4.19 (ABq, J = 14.9 Hz,
2H), 4.49 (s, 2H), 7.33 (d, J = 8.4 Hz, 1H), 7.48−7.55 (m, 1H), 7.66−
7.72 (m, 1H). HRMS calculated for C15H14Cl2N3O: 322.0514. Found:
322.0512.
Ethyl 3-(Aminomethyl)-4-(2,4-dichlorophenyl)-2-methyl-5-
oxo-5H-pyrrolo[3,4-b]pyridine-6(7H)-carboxylate, TFA Salt
(1g). A mixture of 1f (47 mg, 0.096 mmol), 1 M Boc2O/THF (0.18 mL,
0.18 mmol), NaHCO3 (160 mg, 1.9 mmol), and THF (5 mL) was
stirred at room temperature overnight. The mixture was taken up in
EtOAc and H2O and transferred to a separatory funnel. The aqueous
layer was extracted with EtOAc (×2), washed with brine, dried over
Na2SO4, filtered, and evaporated to yield the crude product which was
used as such for the next step without purification. m/z = 422 [M + H]+.
To a solution of the above crude product in CH2Cl2 (5 mL) was
added Et3N (0.1 mL, 0.72 mmol) followed by EtOCOCl (0.04 mL,
0.42 mmol), and the mixture was stirred at room temperature overnight.
LC indicated ∼10% conversion. DMAP (10 mg) followed by additional
EtOCOCl (0.1 mL, 1.05 mmol) was added and mixture allowed to stir at
room temperature overnight. Evaporation and flash chromatography
(40 g silica, 0−100% EtOAc/hexanes) yielded the carbamate (18.5 mg,
39% two steps) as a colorless oil. m/z = 494 [M + H]+. To a solution of
the carbamate (18 mg, 0.046 mmol) in CH2Cl2 (1 mL) was added TFA
(1 mL) dropwise and the mixture stirred at room temperature for 1 h.
Mixture was evaporated and purified by PrepHPLC (Phenomenex
LUNA 5 μm C18(2) 21.2 mm × 100 mm; 8 min gradient; 0−100% B;
20 mL/min) to yield 1g (16.8 mg, 91%) as a white powder. Analytical
HPLC: tR = 5.2 min.
(S)-3-(Aminomethyl)-4-(2,4-dichlorophenyl)-2-methyl-6-(1-
methyl-1H-pyrazol-3-yl)-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-
5-one, TFA Salt (2b). 1H NMR (400 MHz, CD3OD) δ 2.86 (s, 3H),
3.86 (s, 3H), 3.97 and 4.20 (ABq, J = 14.5 Hz, 2H), 4.98 (br s, 2H), 6.65
(d, J = 2.2 Hz, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.52 (d, J = 2.6 Hz, 1H),
7.55 (dd, J = 8.4, 2.2 Hz, 1H), 7.73 (d, J = 1.8 Hz, 1H). HRMS calculated
for C19H18Cl2N5O: 402.0888. Found: 402.0891 [M + H]+.
(S)-3-(Aminomethyl)-4-(2,4-dichlorophenyl)-2-methyl-6-((1-
methyl-1H-pyrazol-3-yl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-
b]pyridin-5-one, TFA Salt (2c). 1H NMR (400 MHz, CD3OD) δ 2.82
(s, 3H), 3.34 (s, 2H), 3.85 (s, 3H), 3.95 and 4.19 (ABq, J = 14.1 Hz, 2H),
4.66 and 4.72 (ABq, J = 15.2 Hz, 2H), 6.18 (d, J = 2.2 Hz, 1H), 7.35 (d,
J = 8.4 Hz, 1H), 7.48−7.57 (m, 1H), 7.70 (d, J = 2.2 Hz, 1H). HRMS
calculated for C20H20Cl2N5O2: 416.1045. Found: 416.1046 [M + H]+.
(S)-3-(Aminomethyl)-4-(2,4-dichlorophenyl)-2-methyl-6-(ox-
azol-2-ylmethyl)-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one,
TFA Salt (2d). 1H NMR (500 MHz, CD3OD) δ 2.74 (s, 3 H), 3.86 and
4.10 (ABq, J = 14.3 Hz, 2 H), 4.53 and 4.57 (ABq, J = 19.0 Hz, 2 H), 4.75
and 4.83 (ABq, J = 16.5 Hz, 2 H), 7.03 (s, 1 H), 7.25 (d, J = 8.3 Hz, 1 H),
7.41 (dd, J = 8.3, 2.20 Hz, 1 H), 7.59 (d, J = 1.7 Hz, 1 H), 7.79 (s, 1 H).
HRMS calculated for C19H17Cl2N4O2: 403.0729. Found: 403.0715
[M + H]+.
(S)-3-(Aminomethyl)-4-(2,4-dichlorophenyl)-2-methyl-6-((5-
methylisoxazol-3-yl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-b]-
pyridin-5-one, TFA Salt (2e). 1H NMR (400 MHz, CD3OD) δ 2.38
(s, 3H), 2.82 (s, 3H), 3.94 and 4.18 (ABq, J = 14.5 Hz, 2H), 4.57 (s, 2H),
4.72 and 4.78 (ABq, J = 15.8 Hz, 2H), 6.08 (s, 1H), 7.32 (d, J = 8.3 Hz,
1H), 7.53 (dd, J = 8.3, 2.0 Hz, 1H), 7.71 (d, J = 2.0 Hz, 1H). HRMS,
Anal. Calcd for C20H19Cl2N4O2: 417.0885. Found: 417.0869 [M + H]+.
(S)-3-(Aminomethyl)-4-(2,4-dichlorophenyl)-6-(2-methox-
yethyl)-2-methyl-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one,
TFA Salt (2f). 1H NMR (400 MHz, CD3OD) δ 2.83 (s, 3H), 3.34 (s,
3H), 3.60 (t, J = 5.3 Hz, 2H), 3.65−3.80 (m, 2H), 3.95 and 4.18 (ABq, J =
14.5 Hz, 2H), 4.62 (s, 2H), 7.33 (d, J = 8.4 Hz, 1H), 7.52 (dd, J = 8.1,
2.0 Hz, 1H), 7.69 (d, J = 2.2 Hz, 1H). HRMS calculated for C18H20Cl2N3O2:
380.0933. Found: 380.0929 [M + H]+. Stereochemistry assigned by
X-ray cocrystal structure of 1e with DPP4.12c It is presumed that the
more active atropisomer (2f) cocrystallized with the protein under the
crystallization conditions.
(S)-3-(Aminomethyl)-4-(2,4-dichlorophenyl)-2-methyl-6-
(((R)-tetrahydrofuran-2-yl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-
3-(Aminomethyl)-4-((S)-2,4-dichlorophenyl)-6-((S)-1-me-
1
b]pyridin-5-one, TFA Salt (2h). H NMR (400 MHz, CD3OD) δ
thoxypropan-2-yl)-2-methyl-6,7-dihydropyrrolo[3,4-b]pyridin-
1
1.53−1.65 (m, 1H), 1.83−1.94 (m, 2H), 1.95−2.06 (m, 1H), 2.83 (s,
3H), 3.52 (dd, J = 14.1, 7.9 Hz, 1H), 3.66−3.76 (m, 2H), 3.82−3.90 (m,
1H), 3.95 and 4.18 (ABq, J = 14.5 Hz, 2H), 4.06−4.15 (m, 1H), 4.60 and
4.71 (ABq, J = 18.9 Hz, 2H), 7.33 (d, J = 7.91 Hz, 1H), 7.52 (dd, J = 8.1,
2.0 Hz, 1H), 7.69 (d, J = 1.8 Hz, 1H). HRMS calculated for C20H22-
Cl2N3O2: 406.1089. Found: 406.1085 [M + H]+. [α]D25 −15.08° (c 1.9,
EtOH).
5-one, TFA Salt (2g). H NMR (400 MHz, CD3OD) δ 1.26 (d, J =
7.0 Hz, 3H), 2.83 (s, 3H), 3.32 (s, 3H), 3.49 (dd, J = 10.1, 4.4 Hz, 1H),
3.59 (dd, J = 12.1, 7.9 Hz, 1H), 3.95 and 4.18 (ABq, J = 14.5 Hz, 2H),
4.52−4.54 (m, 1H), 4.50 and 4.55 (ABq, J = 18.9 Hz, 2H), 7.32 (d, J =
8.4 Hz, 1H), 7.52 (dd, J = 8.4, 1.8 Hz, 1H), 7.70 (d, J = 2.2 Hz, 1H). HRMS
calculated for C19H22Cl2N3O2: 394.1089. Found: 394.1104 [M + H]+.
3-(Aminomethyl)-4-((R)-2,4-dichlorophenyl)-6-((S)-1-me-
thoxypropan-2-yl)-2-methyl-6,7-dihydropyrrolo[3,4-b]pyridin-
(S)-3-(Aminomethyl)-4-(2,4-dichlorophenyl)-2-methyl-6-
1
(((S)-tetrahydrofuran-2-yl)methyl)-6,7-dihydro-5H-pyrrolo[3,4-
5-one, TFA Salt (3b). H NMR (400 MHz, CD3OD) δ 1.27 (d, J =
1
b]pyridin-5-one, TFA Salt (2i). H NMR (400 MHz, CD3OD) δ
7.0 Hz, 3H), 2.83 (s, 3H), 3.31 (s, 3H), 3.49 (dd, J = 10.1, 4.4 Hz, 1H),
3.59 (dd, J = 10.1, 7.9 Hz, 1H), 3.94 and 4.18 (ABq, J = 14.5 Hz, 2H),
4.46−4.55 (m, 1H), 4.48 and 4.57 (ABq, J = 18.5 Hz, 2H), 7.33 (d, J =
8.4 Hz, 1H), 7.52 (dd, J = 8.4, 2.2 Hz, 1H), 7.70 (d, J = 2.2 Hz, 1H). LCMS:
394.10 [M + H]+. HRMS calculated for C19H22Cl2N3O2: 394.1089.
Found: 394.1073 [M + H]+.
(R)-2-(3-(Aminomethyl)-4-(2,4-dichlorophenyl)-2-methyl-5-
oxo-5H-pyrrolo[3,4-b]pyridin-6(7H)-yl)-N,N-dimethylaceta-
mide, TFA Salt (3e). The pure enantiomer was obtained by super-
critical fluid chromatography (chiralpak AD-H 250 mm × 30 mm i.d.;
5 μm; flow rate, 120 mL/min; mobile phase, CO2/EtOH 75/25 (with
0.1%DEA)). This product was further purified by prep HPLC
(Phenomenex Luna 5 μm C18, 21.2 mm × 100 mm; 10 min gradient
from 85% A/15% B to 0% A/100% B (A = 90% H2O/10% MeOH +
0.1% TFA) (B = 90% MeOH/10% H2O + 0.1% TFA); detection at
220 nm) to give 3e as white solid. 1H NMR (400 MHz, CD3OD) δ 2.83
(s, 3H), 2.94 (s, 3H), 3.07 (s, 3H), 3.95 and 4.19 (ABq J = 18.0 Hz, 2H),
4.38 and 4.53 (ABq J = 18.0 Hz, 2H), 4.58 (s, 2H), 7.33 (d, J = 8.3 Hz,
1H), 7.52 (dd, J = 8.3, 2.2 Hz, 1H), 7.69 (d, J = 2.2 Hz, 1H). HRMS:
calculated for C19H20Cl2N4O2: 406.0963. Found: 407.1032 [M + H]+.
1.53−1.65 (m, 1H), 1.83−1.94 (m, 2H), 1.95−2.06 (m, 1H), 2.83 (s,
3H), 3.49 (dd, J = 14.5, 7.7 Hz, 1H), 3.66−3.76 (m, 2H), 3.82−3.90 (m,
1H), 3.95 and 4.19 (ABq, J = 14.5 Hz, 2H), 4.06−4.15 (m, 1H), 4.63 and
4.71 (ABq, J = 18.9 Hz, 2H), 7.34 (d, J = 7.91 Hz, 1H), 7.51 (dd, J = 8.4,
1.8 Hz, 1H), 7.68 (d, J = 2.2 Hz, 1H). HRMS calculated for C20H22-
Cl2N3O2: 406.1089. Found: 406.1092 [M + H]+. [α]D25 + 10.27° (c 2.2,
EtOH).
(S)-3-(Aminomethyl)-4-(2,4-dichlorophenyl)-2-methyl-6-((S)-
pyrrolidin-2-ylmethyl)-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-
5-one, TFA Salt (2j). A mixture of 11c (28 mg, 0.055 mmol) and TFA
(0.7 mL) in CH2Cl2 (1.0 was allowed to stir at ambient temperature for
1 h and evaporated under reduced pressure. The residue was purified by
preparative HPLC (Luna 5 μm C18 (2), 30 mm × 100 mm, 10 min
gradient, 0−60% solvent B, 40 mL/min) to give 2j (15.9 mg, 45.7%
yield) as an off-white solid. 1H NMR (400 MHz, CD3OD) δ 1.59−1.75
(m, 1 H), 1.83−2.05 (m, 2 H), 2.07−2.18 (m, 1 H), 2.75 (s, 3 H), 3.10−
3.30 (m, 2 H), 3.64−3.98 (m, 4 H), 4.12 (part of ABq, J = 14.1 Hz, 1 H),
4.50 and 4.61 (ABq, J = 18.0 Hz, 2 H), 7.26 (d, J = 8.4 Hz, 1 H), 7.42 (dd,
J = 8.1, 2.0 Hz, 1 H), 7.60 (d, J = 2.20 Hz, 1 H). HRMS calculated
K
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Journal of Medicinal Chemistry
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for C20H23Cl2N4O: 405.1249. Found: 405.1256 [M + H]+. Analytical
HPLC: tR = 1.4 min.
resin (5.07 g, 7.5 mmol) in CH2Cl2 (80 mL) at ambient temperature
was added trichloroacetonitrile (0.75 mL, 7.5 mmol) dropwise. The mi-
xture was stirred at ambient temperature for 2.5 h, and additional
triphenylphosphine resin (0.33 g, 0.5 mmol) and trichloroacetonitrile
(50 μL, 0.5 mmol) were added. After 30 min, the mixture was filtered
and the filtrate was concentrated in vacuo to give an acid chloride
(1.12 g) as a light yellow solid. To a solution of the above acid chloride
(0.93 g, 1.98 mmol) in THF (30 mL) at 0 °C was added lithium tri-tert-
butoxyaluminohydride dropwise during a period of 5 min. The mixture
was stirred at 0 °C for 20 min, quenched with H2O (1.3 mL), and con-
centrated in vacuo. Purification of the residue by flash chromatography
(40 g column, 0−100% EtOAc/hexanes) afforded 16 (690.4 mg, 80%
for 2 steps) as an off-white solid. 1H NMR (500 MHz, CD3OD) δ 1.45
(s, 9 H), 2.84 (s, 3 H), 4.20 and 4.26 (ABq, J = 17.6, 2 H), 4.32 and 4.58
(ABq, J = 12.1 Hz, 2 H), 4.56 (s, 2 H), 7.32 (d, J = 8.3 Hz, 1 H), 7.44 (d,
J = 8.3 Hz, 1 H), 7.59 (s, 1 H). m/z = 437 [M + H]+.
(R)-tert-Butyl 2-(3-(Chloromethyl)-4-(2,4-dichlorophenyl)-2-
methyl-5-oxo-5H-pyrrolo[3,4-b]pyridin-6(7H)-yl)acetate (17).
16 (5.7 g) was separated by supercritical fluid chromatography (Chiralpak
AD 250 mm × 4.6 mm i.d.; 10 μm; 35 °C; flow rate, 2.0 mL/min; mobile
phase, CO2/IPA 82/18; injection volume, 5 μL; detector wavelength,
220 nm) to give isomer A (2.43 g, tR = 5.1 min, 100% ee) and isomer B
(2.55 g, tR = 7.1 min, 100% ee) as an off-white solid. To a mixture of
isomer B (763 mg, 1.74 mmol) and Et3N (0.97 mL, 6.98 mmol) in
CH2Cl2 (15 mL) at 0 °C was added mesyl chloride (0.41 mL, 5.24 mmol)
dropwise. The stirred mixture was kept at ambient temperature overnight
and evaporated under reduced pressure. The residue was partitioned
between EtOAc and H2O, and the aqueous layer was extracted further
with EtOAc (2×). The combinedorganic extracts were washed with brine,
dried (Na2SO4), and concentrated in vacuo. The crude product was
chromatographed (40 g column, 0−100% EtOAc/hexanes) to give 17
(785.7 mg, 99%) as a white solid. 1H NMR (500 MHz, CDCl3) δ 1.45 (s,
9 H), 2.86 (s, 3 H), 4.14 and 4.29 (ABq, J = 17.6, 2 H), 4.31 (part of ABq,
J = 7.1, 1 H), 4.52−4.60 (m, 3 H), 7.27 (d, J = 8.3, 1 H), 7.39 (dd, J = 8.3,
2.2 Hz, 1 H), 7.54 (d, J = 1.7, 1 H). m/z = 455 [M + H]+.
(S)-2-(3-((tert-Butoxycarbonylamino)methyl)-4-(2,4-dichlor-
ophenyl)-2-methyl-5-oxo-5H-pyrrolo[3,4-b]pyridin-6(7H)-yl)-
acetic Acid (18). A solution of 17 (784 mg, 1.72 mmol) in 7 M NH3 in
MeOH (140 mL) was heated at 50 °C for 50 min, cooled, and concen-
trated to give crude (S)-tert-butyl 2-(3-(aminomethyl)-4-(2,4-dichlor-
ophenyl)-2-methyl-5-oxo-5H-pyrrolo[3,4-b]pyridin-6(7H)-yl)acetate
(1.81 g) as a light orange sticky solid. To the above material in CH2Cl2
(8.0 mL) was added TFA (5.0 mL), and the resulting mixture was
allowed to stir at ambient temperature for 3 h and evaporated under
reduced pressure. The residue was coevaporated with ethanol several
times to give crude (S)-2-(3-(aminomethyl)-4-(2,4-dichlorophenyl)-
2-methyl-5-oxo-5H-pyrrolo[3,4-b]pyridin-6(7H)-yl)acetic acid as a yellow
solid. The yellow solid was dissolved in THF (35 mL) and treated with
saturated aqueous NaHCO3 (20 mL) followed by di-tert-butyl
dicarbonate (1.28 g, 5.85 mmol). The mixture was stirred at ambient
temperature for 2.5 h, and then the pH of the mixture was adjusted to
3 with 1 N HCl. The aqueous layer was extracted with EtOAc (2×), and
the combined organic extracts were washed with brine, dried (Na2SO4),
and evaporated under reduced pressure to give crude 18 (1.375 g) as a
light yellow solid which was used for the next step without further
purification. m/z = 480 [M + H]+.
(S)-3-(3-(Aminomethyl)-4-(2,4-dichlorophenyl)-2-methyl-5-
oxo-5H-pyrrolo[3,4-b]pyridin-6(7H)-yl)propanoic Acid, TFA Salt
(2m). 1H NMR (400 MHz, CD3OD) δ 2.67 (t, J = 6.8 Hz, 2H), 2.83 (s,
3H), 3.73−3.85 (m, 2H), 3.94 and 4.18 (ABq, J = 14.5 Hz, 2H), 4.62 (s,
2H), 7.32 (d, J = 8.4 Hz, 1H), 7.52 (dd, J = 8.1, 2.0 Hz, 1H), 7.70 (d, J =
1.8 Hz, 1H). HRMS calculated for C18H18Cl2N3O3: 394.0725. Found:
394.0735 [M + H]+.
(S)-3-(3-(Aminomethyl)-4-(2,4-dichlorophenyl)-2-methyl-5-
oxo-5H-pyrrolo[3,4-b]pyridin-6(7H)-yl)-N,N-diethylpropana-
1
mide, TFA Salt (2o). H NMR (400 MHz, CD3OD) δ 1.06 (t, J =
7.0 Hz, 3H), 1.14 (t, J = 7.0 Hz, 3H), 2.54−2.80 (m, 2H), 2.83 (s, 3H),
3.32−3.38 (m, 4H), 3.82 (t, J = 6.8 Hz, 2H), 3.94 and 4.18 (ABq, J =
14.5 Hz, 2H), 4.60 and 4.65 (ABq, J = 18.9 Hz, 2H), 7.32 (d, J = 8.40 Hz,
1H), 7.51 (dd, J = 8.1, 2.0 Hz, 1H), 7.69 (d, J = 1.8 Hz, 1H). HRMS
calculated for C22H27Cl2N4O2: 449.1511. Found: 449.1506 [M + H]+.
(R)-2-((S)-3-(Aminomethyl)-4-(2,4-dichlorophenyl)-2-methyl-
5-oxo-5H-pyrrolo[3,4-b]pyridin-6(7H)-yl)-N,N-diethylpropana-
mide, TFA Salt (2p). 1H NMR (400 MHz, CD3OD) possible rotamers;
data for major rotamer: δ 1.09 (t, J = 7.3 Hz, 3H), 1.20 (t, J = 7.0 Hz,
3H), 1.52 (d, J = 7.0 Hz, 3H), 2.84 (s, 3H), 3.18−3.28 (m, 4H), 3.93 and
4.20 (ABq, J = 14.5 Hz, 2H), 4.67 and 4.80 (ABq, J = 18.0 Hz, 2H), 5.24
(q, J = 7.0 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.52 (dd, J = 8.4, 2.2 Hz,
1H), 7.69 (d, J = 2.2 Hz, 1H). HRMS calculated for C22H27Cl2N4O2:
449.1511. Found: 449.1524 [M + H]+.
(S)-3-(Aminomethyl)-6-(((S)-1-(cyclopropylsulfonyl)-
pyrrolidin-2-yl)methyl)-4-(2,4-dichlorophenyl)-2-methyl-6,7-
dihydro-5H-pyrrolo[3,4-b]pyridin-5-one, TFA Salt (2k). 1H NMR
(500 MHz, CD3OD) δ 0.81−0.97 (m, 4 H), 1.65−1.74 (m, 1 H), 1.81−
1.93 (m, 2 H), 1.91−2.03 (m, 1 H), 2.33−2.50 (m, 1 H), 2.73 (s, 3 H),
3.24−3.31 (m, 1 H), 3.34−3.61 (m, 3 H), 3.85 (part of ABq, J = 14.3 Hz,
1 H), 4.06−4.18 (m, 2 H), 4.50−4.66 (m, 2 H), 7.24 (d, J = 8.3 Hz, 1 H),
7.42 (d, J = 8.3 Hz, 1 H), 7.59 (s, 1 H). HRMS calculated for
C23H27Cl2N4O3S: 509.1181. Found: 509.1190 [M + H]+.
(R)-3-(Aminomethyl)-4-(2,4-dichlorophenyl)-6-(2-methox-
yethyl)-2-methyl-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one,
TFA Salt (3c). 1H NMR (400 MHz, CD3OD) δ 2.83 (s, 3H), 3.34 (s,
3H), 3.60 (t, J = 5.1 Hz, 2H), 3.65−3.80 (m, 2H), 3.95 and 4.19 (ABq, J =
14.5 Hz, 2H), 4.62 (s, 2H), 7.33 (d, J = 8.4 Hz, 1H), 7.52 (dd, J = 8.4,
2.2 Hz, 1H), 7.69 (d, J = 1.8 Hz, 1H). HRMS calculated for C18H20-
Cl2N3O2: 380.0933. Found: 380.0931 [M + H]+.
Benzyl 6-(2-tert-Butoxy-2-oxoethyl)-4-(2,4-dichlorophenyl)-
2-methyl-5-oxo-6,7-dihydro-5H-pyrrolo[3,4-b]pyridine-3-car-
boxylate (9h). A mixture of 8b (10.59 g, 21.43 mmol), glycine
tert-butyl ester hydrochloride (8.26 g, 49.29 mmol), and triethylamine
(8.94 mL, 64.29 mmol) in N,N-dimethylacetamide (200 mL) was
heated to 100 °C for 2 h and then cooled to ambient temperature. The
resulting mixture was partitioned between EtOAc and H2O, and the
aqueous layer was extracted further with EtOAc (2×). The combined
organic extracts were washed with H2O and brine, dried (Na2SO4), and
evaporated under reduced pressure. The residue was purified by flash
chromatography (330 g column, 0−100% EtOAc/hexanes) to give 9h
(9.34 g, 77% yield) as a light yellow solid. 1H NMR (500 MHz, CDCl3)
δ 1.44 (s, 9 H), 2.74 (s, 3 H), 4.13 and 4.29 (ABq, J = 17.6 Hz, 2 H), 4.56
(s, 2 H), 5.05 and 5.07 (ABq, J = 11.8, 2 H), 7.00 (d, J = 8.3 Hz, 1 H),
7.06−7.16 (m, 2 H), 7.21−7.40 (m, 5 H). m/z = 541 [M + H]+.
6-(2-tert-Butoxy-2-oxoethyl)-4-(2,4-dichlorophenyl)-2-meth-
yl-5-oxo-6,7-dihydro-5H-pyrrolo[3,4-b]pyridine-3-carboxylic
Acid (15). A mixture of 9h (9.33 g, 17.23 mmol) and 10% palladium on
carbon (1.36 g) in EtOAc (150 mL) was stirred in the presence of hydrogen
(balloon) at ambient temperature for 2 h. The reaction mixture was filtered
through a pad of Celite and washed with MeOH and CH2Cl2. The filtrate
was evaporated under reduced pressure to give crude 15 (8.13 g, 100%
yield) as a dark yellow solid. 1H NMR (400 MHz, CD3OD) δ 1.45 (s, 9 H),
2.75 (s, 3 H), 4.23 and 4.28 (ABq, J = 18.0 Hz, 2 H), 4.63 and 4.58 (ABq, J =
18.0 Hz, 2 H), 7.27 (d, J = 8.4 Hz, 1 H), 7.39 (dd, J = 8.1, 2.0 Hz, 1 H), 7.55
(d, J = 2.2 Hz, 1 H). m/z = 451 [M + H]+.
2-(3-((Bis-tert-butoxycarbonylamino)methyl)-4-(2,4-dichlor-
ophenyl)-2-methyl-5-oxo-5H-pyrrolo[3,4-b]pyridin-6(7H)-yl)-
N,N-dimethylacetamide (19). To a stirred solution of 18 (8.28 g,
19.4 mmol) in THF (125 mL) at 60 °C was added di-tert-butyl imi-
nodicarboxylate, potassium salt (9.95 g, 9.40 mmol). DMF (41.5 mL)
was added and the reaction mixture heated at 60 °C for 5 min and stirred
at room temperature for another 25 min. The reaction mixture was
concentrated under reduced pressure to remove THF. The resulting
residue was diluted with EtOAc (570 mL) and extracted with 5% LiCl
solution (2 × 380 mL) and brine. The aqueous layers were extracted
with EtOAc (1 × 400 mL, 1 × 200 mL). The combined organic layers
were dried (MgSO4), filtered, and concentrated to give a yellow foam
which was purified by silica gel chromatography (EtOAc) to give the
tert-Butyl 2-(4-(2,4-Dichlorophenyl)-3-(hydroxymethyl)-2-
methyl-5-oxo-5H-pyrrolo[3,4-b]pyridin-6(7H)-yl)acetate (16).
To a stirred mixture of 15 (1.18 g, 2.5 mmol) and triphenylphosphine
1
desired product (7.19 g, 60%) as a white solid. H NMR (400 MHz,
L
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Article
CDCl3) δ 1.36 (s, 18H), 2.81 (s, 3H), 2.93 (s, 3H), 3.00 (s, 3H), 4.22
and 4.46 (ABq, JAB = 16.5 Hz, 2H), 4.56 and 4.69 (ABq, JAB = 17.3 Hz,
2H), 4.66 and 4.91 (ABq, JAB = 15.2 Hz, 2H), 7.23 (d, J = 8.3 Hz, 1H),
7.30 (dd, J = 8.3, 2.2 Hz, 1H), 7.45 (d, J = 2.2 Hz, 1H). Analytical HPLC:
tR = 8.60 min. m/z = 607 [M + H]+.
(S)-2-(3-((Bis-tert-butoxycarbonylamino)methyl)-4-(2,4-di-
chlorophenyl)-2-methyl-5-oxo-5H-pyrrolo[3,4-b]pyridin-6(7H)-
yl)-N,N-dimethylacetamide (20). The desired atropisomer (slower-
eluting) was obtained by chiral SFC separation (Chiralpak AD 250 mm ×
4.6 mm i.d.; 10 μm; 35 °C; flow rate, 2.0 mL/min; mobile phase, CO2/
IPA 82/18; injection volume, 5 μL; detector wavelength, 220 nm) in
96% recovery and >99% ee.
161.22, 143.16, 135.09, 133.70, 133.41, 132.08, 130.80, 129.29, 127.04,
121.22, 51.88, 43.27, 39.83, 36.35, 35.60, 23.04. HRMS calculated
for C19H21N4O2Cl2: 407.1042. Found: 407.1041. Elemental analysis
calculated: C 55.80, H 4.99, N 13.65, Cl 17.27. Found: C 56.09, H 4.71,
N 13.73, Cl 17.40. [α]D +18.8 (c 1.024, MeOH). Analytical HPLC:
tR = 2.6 min.
25
(S)-2-(3-(Aminomethyl)-4-(2,4-dichlorophenyl)-2-methyl-5-
oxo-5H-pyrrolo[3,4-b]pyridin-6(7H)-yl)-N,N-diethylacetamide,
TFA Salt (2n). 59% yield over two steps. 1H NMR (500 MHz, CD3OD)
δ 1.12 (t, J = 7.2 Hz, 3 H), 1.24 (t, J = 7.2 Hz, 3 H), 2.84 (s, 3 H), 3.34−
3.48 (m, 4 H), 3.96 and 4.20 (ABq, J = 14.3 Hz, 2 H), 4.40 and 4.52 (ABq,
J = 16.7 Hz, 2 H), 4.61 (s, 2 H), 7.34 (d, J = 8.3 Hz, 1 H), 7.51 (d, J =
8.3 Hz, 1 H), 7.69 (s, 1 H). HRMS calculated for C21H25Cl2N4O2:
435.1355. Found: 435.1366 [M + H]+.
(S)-2-(3-(Aminomethyl)-4-(2,4-dichlorophenyl)-2-methyl-5-
oxo-5H-pyrrolo[3,4-b]pyridin-6(7H)-yl)-N-methylacetamide,
TFA Salt (2r). 74% yield over two steps. 1H NMR (400 MHz, CD3OD)
δ 2.63 (s, 3 H), 2.74 (s, 3 H), 3.86 and 4.11 (ABq, J = 14.5 Hz, 2 H), 4.08
and 4.20 (ABq, J = 16.8 Hz, 2 H), 4.50 (s, 2 H), 7.25 (d, J = 8.3 Hz, 1 H),
7.42 (dd, J = 8.3, 1.6 Hz, 1 H), 7.59 (d, J = 1.6 Hz, 1 H). HRMS
calculated for C18H19Cl2N4O2: 393.0885. Found: 393.0894 [M + H]+.
(S)-2-(3-(Aminomethyl)-4-(2,4-dichlorophenyl)-2-methyl-5-
oxo-5H-pyrrolo[3,4-b]pyridin-6(7H)-yl)-N-methyl-N-propylace-
tamide, TFA Salt (2t). 57% yield over two steps.1H NMR (500 MHz,
CD3OD) δ 0.88 and 0.96 (t, J = 7.3 Hz, 3 H, rotamers), 1.56 and 1.68 (q,
J = 7.2 Hz, 2 H, rotamers), 2.84 (s, 3 H), 2.92 and 3.06 (s, 3 H, rotamers),
3.26−3.41 (m, 2 H), 3.96 and 4.20 (ABq, J = 14.3 Hz, 2 H), 4.33−4.61
(m, 4 H), 7.34 (d, J = 7.7 Hz, 1 H), 7.51 (d, J = 8.3 Hz, 1 H), 7.68 (s, 1H).
HRMS calculated for C21H25Cl2N4O2: 435.1355. Found: 435.1368
[M + H]+.
(S)-2-(3-(Aminomethyl)-4-(2,4-dichlorophenyl)-2-methyl-5-
oxo-5H-pyrrolo[3,4-b]pyridin-6(7H)-yl)acetamide, TFA Salt
(2q). To a mixture of 18 (70 mg, 0.146 mmol), HOBT·H2O (29.6 mg,
0.218 mmol), and ammonium chloride (15.6 mg, 0.292 mmol) in DMF
(0.5 mL) were added EDCI (42 mg, 0.218 mmol) and diisopropylethyl-
amine (0.101 mL, 0.58 mmol). The mixture was stirred at ambient
temperature overnight and then diluted with EtOAc (10 mL) and H2O
(2.5 mL). The organic layer was separated and evaporated in vacuo to
yield the crude amide product. To a solution of the amide product in
CH2Cl2 (1 mL) was added TFA (0.5 mL), and the resulting mixture was
stirred for 3 h. Solvent was removed in vacuo and the residue purified by
preparative HPLC (Phenomenex Axia, 8 min gradient, 0−100% B) to
give 2q (8.6 mg, 12%) as a white powder. 1H NMR (400 MHz, CD3OD)
δ 2.76 (s, 3H), 3.88 and 4.12 (ABq, J = 15.0 Hz, 2H), 4.13 and 4.24 (ABq,
J = 17.2 Hz, 2H), 4.54 (s, 2H), 7.26 (d, J = 8.3 Hz, 1H), 7.45 (dd, J = 8.3,
2.2 Hz, 1H), 7.62 (d, J = 2.2 Hz, 1H). HRMS, Anal. Calcd for C17H17-
Cl2N4O2: 379.0729. Found: 379.0739 [M + H]+. Analytical HPLC: tR =
3.0 min.
(S)-2-(3-(Aminomethyl)-4-(2,4-dichlorophenyl)-2-methyl-5-
oxo-5H-pyrrolo[3,4-b]pyridin-6(7H)-yl)-N,N-dimethylaceta-
mide (2s). Procedure A. To a solution of 18 (100.9 mg, 0.21 mmol) in
THF (5.0 mL) was added benzotriazol-1-yloxytripyrrolidinophospho-
nium hexafluorophosphate (163.9 mg, 0.315 mmol), N,N-diisopropy-
lethylamine (0.15 mL, 0.84 mmol), and 2 M NHMe2/THF (0.157 mL,
0.315 mmol). The reaction mixture was stirred at ambient temperature
for 2.5 h and evaporated. The residue was partitioned between EtOAc
and H2O, and the aqueous layer was extracted further with EtOAc (2×).
The combined organic extracts were washed with H2O and brine, dried
with (Na2SO4), and evaporated under reduced pressure to give the
crude amide (186.1 mg) as a light orange solid. The above crude product
was dissolved in CH2Cl2 (1.5 mL) and treated with TFA (0.8 mL). The
reaction mixture was allowed to stir at ambient temperature for 2.5 h and
concentrated in vacuo. The residue was purified by preparative HPLC
(YMC S5 ODS 30 mm × 100 mm, 12 min gradient, 0−80% solvent B,
40 mL/min) to give 2s, TFA salt (78.9 mg, 98.2% ee, 70% yield over two
steps) as an off-white powder. 1H NMR (500 MHz, CD3OD) δ 2.83 (s,
3 H), 2.94 (s, 3 H), 3.07 (s, 3 H), 3.95 and 4.19 (ABq, J = 14.3 Hz, 2 H),
4.39 and 4.51 (ABq, J = 17.0 Hz, 2 H), 7.33 (d, J = 8.3 Hz, 1 H), 7.51 (d,
J = 8.3 Hz, 1 H), 7.69 (s, 1 H). HRMS calculated for C19H21Cl2N4O2:
407.1042. Found: 407.1046 [M + H]+.
(S)-3-(Aminomethyl)-4-(2,4-dichlorophenyl)-2-methyl-6-(2-
oxo-2-(pyrrolidin-1-yl)ethyl)-6,7-dihydro-5H-pyrrolo[3,4-b]-
1
pyridin-5-one, TFA Salt (2u). 45% yield over two steps. H NMR
(500 MHz, CD3OD) δ 1.83−1.95 (m, 2 H), 1.96−2.09 (m, 2 H), 2.84
(s, 3 H), 3.43 (t, J = 6.9 Hz, 2 H), 3.52 (t, J = 6.9 Hz, 2 H), 3.96 and 4.20
(ABq, J = 14.3 Hz, 2 H), 4.33 and 4.44 (ABq, J = 17.1 Hz, 2 H), 4.62 (s, 2
H), 7.34 (d, J = 8.3 Hz, 1 H), 7.51 (d, J = 8.3 Hz, 1 H), 7.69 (s, 1 H).
HRMS calculated for C21H23Cl2N4O2: 433.1198. Found: 433.1194
[M + H]+.
(S)-3-(Aminomethyl)-4-(2,4-dichlorophenyl)-2-methyl-6-(2-
oxo-2-(piperidin-1-yl)ethyl)-6,7-dihydro-5H-pyrrolo[3,4-b]-
pyridin-5-one, TFA Salt (2v). 60% yield over two steps. 1H NMR (500
MHz, CD3OD) δ 1.55 (m, 2 H), 1.65 (m, J = 24.2, 3.9 Hz, 4 H), 2.84 (s,
3 H), 3.47 (t, J = 5.3, 2H), 3.52 (t, J = 5.0, 2H), 3.96 and 4.20 (ABq, J =
14.6 Hz, 2 H), 4.41 and 4.50 (ABq, J = 16.8 Hz, 2 H), 4.59 (s, 2 H), 7.34
(dd, J = 8.3, 2.2 Hz, 1 H), 7.51 (dd, J = 8.3, 1.7 Hz, 1 H), 7.68 (s, 1 H).
HRMS calculated for C22H25Cl2N4O2: 447.1355. Found: 447.1362
[M + H]+.
In Vitro DPP4 Inhibition Assays. Inhibition of human DPP4
activity was measured under steady-state conditions by following the
absorbance increase at 405 nm upon the cleavage of the pseudosub-
strate, Gly-Pro-pNA. Assays were performed in 96-well plates using
a Thermomax plate reader. Typical reactions contained 100 μL of
ATE buffer (100 mM Aces, 52 mM Tris, 52 mM ethanolamine, pH 7.4),
0.45 nM enzyme, either 120 or 1000 μM substrate (S < Km and S > Km,
Km = 180 μM) and 10 or 11 concentrations of the inhibitor. To ensure
steady-state conditions for slow-binding inhibitors, enzyme was
preincubated with the compound for 40 min prior to substrate addition.
All serial inhibitor dilutions were in DMSO, and final solvent con-
centration did not exceed 1%. Inhibitor potency was evaluated
by calculating IC50 values at each substrate concentration and then
converting them to Ki values by assuming competitive inhibition ac-
cording to the equation Ki = IC50/[1 + (S/Km)]. All inhibitors were
competitive as judged by close agreement of Ki values obtained from
assays at high and low substrate concentrations. In cases where IC50 at
the low substrate concentration was close to the enzyme concentration
used in the assay, the data were fit to the Morrison equation to account
for the depletion of the free inhibitor.
Procedure B. A mixture of 20 (4.4 g, 7.3 mmol) and 4 N HCl in
dioxane (30 mL) was stirred at room temperature for 8.5 h. The reaction
mixture was diluted with ether (150 mL) and filtered. The solid cake
was washed with ether (150 mL) and dissolved in MeOH (60 mL). The
volatiles were removed under reduced pressure to give 2s, dihydr-
ochloride salt, as a beige solid (3.3 g, 86%). The dihydrochloride salt
of 2s thus obtained (5.63 g, 11.6 mmol) was dissolved in CH2Cl2
(100 mL), and 1 N NaOH (75 mL) was added slowly. The resulting
mixture was transferred to a separatory funnel, and the aqueous layer
was extracted with CH2Cl2 (2 × 100 mL). The combined organic
layer was washed with brine, dried (Na2SO4), filtered, and concentrated
under reduced pressure to afford the free base as a foamy solid. Recrys-
tallization from EtOAc yielded 2s, crystalline free base (4.23 g, 90%), as
a colorless solid. Mp 193−194 °C. 1H NMR (400 MHz, methanol-d4)
δ 2.82 (s, 3 H), 2.93 (s, 3 H), 3.06 (s, 3 H), 3.57 and 3.75 (ABq, JAB
=
13.9 Hz, 2 H), 4.39 and 4.48 (ABq, JAB = 16.8 Hz, 2 H), 4.54 (s, 2 H),
7.33 (d, J = 8.2 Hz, 1 H), 7.45 (dd, J = 8.4, 2.0 Hz, 1 H), 7.61 (d, J =
2.0 Hz, 1 H). 13C NMR (125 MHz, CDCl3) δ 167.34, 166.58, 162.39,
Oral Glucose Tolerance Test in ob/ob Mice. Male 13- to 14
week-old ob/ob mice (Jackson Labs) were maintained under constant
temperature and humidity conditions and a 12 h/12 h light−dark cycle
M
dx.doi.org/10.1021/jm4008906 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
and had free access to a 10% fat rodent diet (D1245B Research
Diets) and tap water. After an overnight fasting period, animals were
dosed orally with vehicle (water) or DPP4 inhibitor (0.1−10 μmol/kg)
at −60 min. Two blood samples were collected at −60 and 0 min by tail
bleed for glucose and insulin determinations. Glucose (2 g/kg) was then
administered orally (at 0 min). Additional blood samples were collected
at 15, 30, 60, 90, and 120 min for glucose and insulin determinations.
Blood samples were collected into EDTA containing tubes (Sarstedt).
Plasma glucose was determined with an Accu-Chek Advantage (Roche)
glucometer. Plasma insulin was assayed using a mouse insulin ELISA kit
(ALPCO Diagnostics). Data represent the mean of 12−24 mice/group.
Data analysis was performed using one way ANOVA followed by
Dunnett’s test. All procedures were performed according to BMS-
IACUC guidelines.
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AUTHOR INFORMATION
Corresponding Author
■
*Telephone: 609-818-4974. Fax: 609-818-3550. E-mail:
pratik.devasthale@bms.com.
Present Address
∞L.G.H.: Novartis; e-mail, lawrence.hamann@novartis.com.
#R.Z.: PharmD Consulting, LLC; e-mail, bob@rzahler.com.
Notes
The authors declare no competing financial interest.
×Deceased.
ACKNOWLEDGMENTS
■
We thank our colleagues from Discovery Analytical Sciences for
their assistance in the characterization of the compounds
reported herein, Discovery Synthesis for help with intermediate
synthesis at BMS and Syngene, Javed Khan for help with PDB
X-ray database depositions, and Fang Moore for help with
preparation of the manuscript.
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ABBREVIATIONS USED:
■
hERG EP, human ether-a-go-go related gene; CYP3A4, cyto-
chrome P450 3A4; iv, intravenous; po, per os (oral); oGTT, oral
glucose tolerance test; AUC, area under the curve; PDB, Protein
Data Bank
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O
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